beta1 integrin/c-Met: a receptor complex driving invasive resistance to anti-angiogenic therapy in glioblastoma

 描述(由申请人提供) 恶性肿瘤如胶质母细胞瘤严重依赖血管生成来维持其高度增殖和侵袭性的表型。因此，抗血管生成治疗对于这种极具破坏性的脑癌是非常有希望的，而有效的治疗是非常必要的。不幸的是，人们对抗血管生成疗法(如血管内皮生长因子中和抗体贝伐单抗)的热情已经减退，因为尽管癌症对这种疗法的初始反应通常是深刻的，但许多肿瘤会产生侵袭性耐药，疾病进展迅速导致预后不佳。临床前研究表明，与耐药通常需要基因突变的传统DNA损伤化疗不同，抗血管生成治疗耐药是通过转录重新编程或蛋白质翻译后修饰发生的，允许肿瘤生长复发，尽管抗血管靶点受到抑制。我们假设，上调的β1整合素和受体酪氨酸激酶c-Met通过物理相互作用驱动对抗血管生成治疗的侵袭性抵抗，导致一个结构复合体，其功能不仅是放大它们的信号和相互下游的中介，而且通过激活该复合体的其他特定通路。我们将通过特定的系统来研究这一假设，这些系统通过化学和光刺激在c-Met和β1整合素之间激发蛋白质-蛋白质相互作用(目标1)。利用CRISPRi技术，我们将整合我们蛋白质的双重和接近完全的敲除，通过消除背景复合体来进一步优化诱导复合体的生物影响。该复合体的诱导将通过一系列的体外生物学试验进行分析，并在体内进行后续的抗血管生成治疗的反应评估。此外，我们将利用人磷酸激酶阵列来筛选针对复合体形成的特定通路的激活。为了研究该复合体的破坏是否消除了耐药性，我们首先需要阐明它们的哪个结构域以及这些结构域中的哪些特定残基允许β1整合素和c-蛋氨酸相互结合(目标2)。这将通过使用PCR定点突变将靶向突变引入每个受体，并通过CRISPRi敲除在具有这些受体遗传缺陷的肿瘤细胞中表达这些突变形式来实现。然后将使用免疫沉淀来确认受体结合。仅携带不能结合的β1整合素和c-蛋氨酸的改变版本的肿瘤细胞对抗血管生成治疗的反应可以在 体内原位微环境。这些目标的成功完成将表征β1整合素和c-蛋氨酸复合体形成的生物学效应，阐明这一独特的复合体驱动抗性的机制，并确定这两种蛋白之间的结合部位(S)。该项目的研究结果可能对未来维持抗血管生成治疗效果和延缓耐药性演变的治疗靶点至关重要。

 DESCRIPTION (provided by applicant) Malignant tumors such as glioblastoma rely heavily on angiogenesis to endure sustain their highly proliferative and aggressive phenotype. Anti-angiogenic therapy is therefore very promising for this devastating brain cancer for which effective treatments are greatly needed. Unfortunately, the enthusiasm that greeted the advent of anti-angiogenic therapies such as the VEGF-neutralizing antibody bevacizumab, has been mitigated because, although the initial responses of cancers to this therapy are often profound, many tumors develop invasive resistance, with rapid disease progression leading to poor outcomes. Preclinical studies suggest that, unlike conventional DNA damaging chemotherapy whose resistance often requires gene mutations, anti- angiogenic therapy resistance occurs via transcriptional reprogramming or post-translational modification of proteins, allowing for tumor growth resurgence to take place despite inhibition of the antivascular target. We hypothesize that upregulated β1 integrin and receptor tyrosine kinase c-Met drive invasive resistance to anti- angiogenic therapy through a physical interaction, resulting in a structural complex that functions not only to amplify their signaling va mutual downstream mediators, but through activation of additional pathways specific to this complex. We will investigate this hypothesis through specific systems that evoke protein-protein interaction between c-Met and β1 integrin via both chemical and light stimulation (Aim 1). Utilizing CRISPRi technology, we will incorporate dual and near-complete knockdowns of our proteins to further optimize the biological impact of the induced complex by eliminating background complexes. The induction of this complex will be analyzed through a series of biological assays in vitro and followed up for the assessment of response to anti-angiogenic therapy in vivo. Furthermore, we will screen for the activation of pathways specific to complex formation utilizing a Human Phosphokinase Array. To investigate whether disruption of the complex abrogates resistance, we will first need to elucidate which of their domains and which specific residues within these domains allow β1 integrin and c-Met to bind each other (Aim 2). This will be achieved by introducing targeted mutations into each receptor using PCR site-directed mutagenesis, and expressing these mutant forms in tumor cells rendered genetically deficient in these receptors through CRISPRi knockdown. Receptor binding will then be confirmed using immunoprecipitation. The response to anti-angiogenic therapy of tumor cells carrying only altered versions of β1 integrin and c-Met which fail to bind can then be assessed in the orthotopic microenvironment in vivo. Successful completion of these aims will characterize the biological effects of the β1 integrin and c-Met complex formation, elucidate fundamental insight into the mechanism by which this unique complex drives resistance, and determine the binding site(s) between these two proteins. The findings from this program can prove to be essential for future therapeutic targets to sustain the efficacy of anti-angiogenic therapy and delay the evolution of resistance.